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Abstract

Respiratory syncytial virus (RSV) belongs to the paramyxovirus family that includes many clinically relevant viruses, such as the human metapneumovirus and measles. RSV infection can cause severe disease in infants, the elderly, and some immunocompromised adults. During RSV replication, a series of truncated forms of the viral genome is generated. These truncated viral genomes are known as defective viral genomes (DVGs) and are generated by many viruses (Lazzarini et al., 1981; Rao and Huang, 1982; Prince et al., 1996; Sun et al., 2015; Tapia et al., 2013). DVGs can restrict the replication of the full-length virus and are the primary natural triggers of the innate immune response to RSV (Sun et al., 2015; Tapia et al., 2013). Here we discuss in detail how to prepare RSV stocks with a high or low content of DVGs, and how to purify defective viral particles containing DVGs from an RSV stock enriched in defective viral particles. These procedures are useful for the preparation of viral stocks and defective viral particles necessary for laboratory research. In brief, the different RSV stocks are produced in HEp2 cells, which are commonly used to amplify this virus in the laboratory. To generate an RSV stock with a high content of DVGs, HEp2 cells are sequentially infected with a high multiplicity of infection (MOI) multiple times followed by purification of the viral particles containing DVGs using gradient centrifugation. The procedure describe here has four parts: 1. Amplification of seed RSV stock with a low DVG content (RSV-LD), 2. Generation of a stock with a high DVG content (RSV-HD), 3. Purification of DVGs by gradient centrifugation, 4. Characterization of purified DVGs.

Preparation of an RSV seed stock with a low content of defective viral particles (RSV-LD) Note: This step aims to amplify the original virus purchased from ATCC. The goal is to have enough material to then generate an RSV-HD stock.

For initial amplification of the virus, seed 5 x 105/well
mycoplasma-free HEp2 cells in TCM in a 6-well plate one night before
virus infection. Note: Cells and viral infections in this protocol
are incubated in a tissue culture incubator at 37 °C, 7% CO2 (5% CO2 can
also be used).

Next day, wash the cells twice with sterile PBS
and infect with RSV at an MOI of 0.01 tissue culture infectious dose
(TCID50)/cell. Use the seeded number of cells for MOI calculation.
Dilute the virus in infection medium and use a total volume of 200 μl
per infection. Incubate at 37 °C in a tissue culture incubator for 2 h.

Rock the plates once every 15-20 min to maintain an even virus distribution and avoid the cells drying out.

After 2 h of incubation, add 2 ml infection medium (see Recipes) per well and incubate in a tissue culture incubator.

Harvest the virus 5 days post-infection by scrapping the cells and collecting them together with the culture supernatants.

Centrifuge the pooled cells and supernatants for 5 min at 280 x g in
a table-top centrifuge at 4 °C and separate the supernatant leaving
approx. 200 μl of supernatant in the tube with the pelleted cells.

Resuspend the cell pellet with the leftover 200 μl supernatant and
quick-freeze the mixture in dry ice/ethanol, followed by quick thaw in
a 37 °C water bath. Repeat this quick freeze/thaw step at least 3 times
and vortex after every time.

Pool all the freeze-thawed cell
debris with the saved supernatant, vortex to mix well, aliquot (500
μl/tube), and quick-freeze the virus in dry ice/ethanol. This is Passage
1 virus (P1).

Amplify this P1 virus a second time (P2).

For P2 amplification, seed 3 x 106 HEp2 cells in one T-75 flask and
infect with 200 μl P1 virus diluted in 1 ml infection medium, following
the same procedure as P1. After 2 h of incubation in a tissue culture
incubator add 9 ml of infection medium to each flask.

Harvest the virus 5 days after infection as described for P1. Aliquot (100 μl/vial) and quick-freeze the virus.

Titrate the P2 (containing both supernatants and debris) before
proceeding with the RSV-HD preparation (for titration method please see
TCID50 section below). Note: Cell death should be noticed 4-5 days
post infection (Figure 1). If earlier cell death is noticed, reduce the
amount of P1 virus. If no cell death is noticed post 5 days infection,
passage P2 another time following steps A10-12.

Preparation and concentration of an RSV stock with a high content of defective viral particles (RSV-HD). Note: This step aims to enrich DVGs in the RSV stock. The goal is to have enough DVG-containing defective viral particles to purify from the viral stock. The standard way to promote DVG production during viral infection is to sequentially passage the virus stock (generated from section A in vitro using a high MOI.

To prepare an RSV-HD seed stock, infect HEp2 cells with RSV-LD P2 at a
moi of 4 TCID50/cell for two passages to generate RSV-HD P1 and P2. Use
the same procedure as for LD preparation. Specifically, T75 flasks are
used for RSV-HD P1 and P2 amplification with an inoculum of 1 ml/flask.
Additional 9 ml of infection medium should be added after 2 h of
incubation. Calculate the viral titers using the TCID50 assay (see
TCID50 section).

Harvest viruses 2 days post-infection or when
more than half of the cells are dead. Quick freeze/thaw the cell pellet
at least 3 times as described in steps A6-7.

Collect the supernatants and discard the cell debris. Aliquot (500 μl/vial) and quick-freeze the viruses.

To generate an RSV-HD working stock, seed 107 HEp2 cells in one
T-225 flask and infect with RSV-HD P2 at a moi of 10 TCID50/cell. Use an
inoculum of 4 ml per T-225 flask and follow the infection procedure
described above. Add 21 ml of infection medium after 2 h incubation of
viruses and put it back in the tissue culture incubator.

Two
days later, or when more than half of the cells have detached from the
plate, scrape the cells and collect them together with the culture
supernatant to harvest the virus.

Centrifuge for 5 min at 280 x g in a table-top centrifuge at 4 °C and collect the supernatant.

Return 2-3 ml of the supernatant to each pellet, and put the rest
of the supernatants on ice. Suspend the pellet, vortex, and quick
freeze/thaw the mixtures at least 3-4 times as in steps A6-7.

Centrifuge for 5 min at 280 x g in a table-top centrifuge, the goal is
to eliminate debris. Take the supernatants and combine them with the
rest of the supernatants. Caution: Discard the debris at this step.

Ultra-centrifuge the supernatants for 2.5 h at 59,000 x g at 4 °C
using an SW 32 Ti Rotor and Ultra-ClearTM 25 x 89 mm centrifuge tube to
concentrate the RSV-HD working stock.

Lightly wash with 1 ml of HBSS twice without disturbing the pellet.

Suspend the pellet in a total 200 μl of infection medium / T225 flask.

Aliquot (200 μl /vial) and quick-freeze the viruses in dry ice/ethanol for storage.

In order to obtain enough defective viral particles, we usually
infect 48 T225-flasks, totaling 9 ml of ultra-centrifuged RSV-HD (less
virus may be sufficient but the yield decreases).

Purification of RSV defective viral particles Note: This step aims to isolate the defective viral particles containing DVGs from the infectious viral particles with full-length genome. Since defective viral particles are much smaller and less dense compared to standard viral particles, the lighter (in our case is the top) fraction contains the most DVGs.

Make 100 ml of PNE buffer fresh in the biosafety hood, shake and mix well.

Fill each tube with 5 ml of 20% sucrose solution. Carefully and
slowly pipette 5 ml 60% sucrose solution starting at the bottom of the
tube and slowly lift up the 20% sucrose layer.Caution: Slow action is critical. A sharp separation between 20% and 60% sucrose solutions should be observed.

Carefully load the tubes containing sucrose solution on the
Gradient Master to generate 20%-60% sucrose gradient. Do this in the
biosafety hood to maintain sterility.

Once finished, carefully
layer 1.5 ml of ultra-centrifuged RSV-HD (prepared in step B) on top of
each sucrose gradient. Use a P1000 micropipette to place the virus. The
tubes should be filled up to 2-3 mm from the edge to avoid them
collapsing during ultracentrifugation. Caution: Do not disturb the sucrose gradient when adding the virus.

Carefully remove the top pink layer once finished the centrifuge. Note: you should be able to observe several cloudy layers. Extract the
cloudy layers from top to bottom using a P1000 micropipette. Transfer
and combine the same fraction from all 6 tubes to a separate clean
Ultra-Clear 14 x 89 mm centrifuge tube. In total, 3 layers are extracted
from RSV-HD, named Fraction 1, 2, 3 from top to bottom (Figure 2).

Fill up each tube to 2-3 mm from the top with PBS and ultra-centrifuge for 2 h at 4 °C at 106,000 x g using SW 41 Ti Rotor.

Suspend the pellet from each fraction in 1 ml of 0.1% Gelatin in
PBS, aliquot 100 μl per vial, snap freeze, and store at -80 °C.

Virus Titration by TCID50 (infectious viral titer) Note: This step aims to quantify the infectious viral particles contained in each of the purified fractions based on their ability to replicate in permissive cells.

The day before titration prepare 96 well plates with HEp2 cells. Seed
the plate with 2 x 104 cells / well in TCM (100 μl /well of a solution
of 2 x 106 cells/ 10 ml TCM). One plate will fit four test samples.
Prepare as many plates as needed.

The day of the titration the cells should be 80-90% confluent.

In a separate 96 well plate, prepare 1/10 serial dilutions of
fractions 1, 2, and 3 in triplicates in infection media. To do this add
90 μl of infection media to all wells in the plate. Add 10 μl of each
fraction to each well on the first row. Prepare triplicates for each
fraction. Leave three rows for media alone, and include a positive
control with a virus of known titer. Use a multichannel pipette to mix
the virus with the infection media in the first row and transfer 10 μl
to the next row. Change tips, mix and transfer 10 μl to the next row,
continue until the last row. Make sure to mix the virus well and change
the tips for every dilution.

Remove the TCM of the plate containing the cells and wash the monolayer twice with 100 μl of serum free media.

Using a multichannel pipette, transfer 25 μl/well of the virus
dilutions to the cells and incubate for 2 h at 37 °C in a tissue culture
incubator. Start transferring from the bottom of the plate (higher
dilution) to avoid carrying over virus from the higher concentrations.

Add 75 μl of infection media per well and incubate for 4-5 days in a
tissue culture incubator. Be careful not to contaminate the media with
virus from the plate.

To determine the viral titer, discard the media in a glass tray containing 10% bleach.

Add 100 μl of crystal violet working solution to the wells. Be careful NOT to touch the bottom of the wells.

Wait for 15-30 min.

Wash the plate by submerging it upside down in water several times to eliminate the excess of crystal violet.

Let it dry at RT.

Score the titer by determining the last dilution with positive CPE
(Figure 3). Score the number of positive wells for that last dilution
(number of positive out of three). From this score calculate the
TCID50/25 μl using the following formula, where “x” correspond to the
dilution:
“+ + +” 10x TCID50 = 10X+0.7/25 μl
“+ + - ” 10x TCID50 = 10X+0.4/25 μl
“+ - - ” 10x TCID50 = 10X-0.1/25 μl

Figure 3. Crystal violet staining of TCID50. The titers from three
different viruses were determined by TCID50 as illustrated above. 10-4 and 10-5 were the last dilutions with positive CPE for designated virus
samples, which were the “x”. “+” stands for the positive CPE observed.
“-” indicates no CPE observed. Based on the equation above, calculate
the final titer of each virus as shown at the bottom of this figure.

Estimation of total amount of viral particles (total virus) Note: This step aims to estimate the quantity of total viral particles present in each of the purified fractions. We use total amount of protein in each fraction measured by Bradford assay as an estimate of total viral particles.

Prepare the BSA standard (provided in the kit) using serial dilutions as shown in Table1.

Table 1. BSA serial dilution for standard curve from 2 mg BSA stock

Std number

Volume of diluent
(μl)

Volume of stock or sample

(μl)

BSA conc.
mg/ml

1

25

75 (BSA: 2 mg/ml)

1.500

2

65

65 (BSA: 2 mg/ml)

1.000

3

35

35 of Std 1

0.750

4

65

65 of Std 2

0.500

5

65

65 of Std 4

0.250

6

65

65 of Std 5

0.125

7

80

20 of Std 6

0.025

Prepare 15 ml of Coomassie blue reagent (provided in the kit) per
96 wells plate and equilibrate it to room temperate before use.

Add 150 μl of Coomassie blue reagent per well to the 96 well plate.

Add 5 μl of the BSA standard and the diluted Fractions 1, 2, and 3.

Mix by tapping the edge of the plate.

Incubate the plate for 15 min at room temperature.

Read O.D at 595 nm using a VariokanTM Flash Multimode Reader or equivalent.

Based on the standard curve, calculate the total protein concentration of Fractions 1, 2, and 3.

Infectivity/Total viral protein ratio calculationNote: This step aims to determine which fraction contains most of the defective viral particles with the least standard infectious viral particles.

The ratio between Infectivity titer (I) and Total viral protein (T) equals to TCID50 per 25 μl / total viral protein per 25 μl.

Select the Fraction with the lowest I/T to further validate the defective viral genome content.

Validation of purified RSV defective viral particles by PCRNote: This step aims to confirm that the fraction selected in section F is enriched in DVGs. For this purpose, permissive cells are incubated with each purified fraction and DVGs are identified using a specific RT-PCR assay (DVG-RT-PCR). For detailed illustration of the DVG RT-PCR assay please refer to Sun et al. (2015), Figure S1.

HEp2 cells are supplemented with either PBS or 1, 10, 20 μl of the
selected Fraction. In a separate well infect with RSV-HD working stock
of MOI of 1 as a control.

Figure 4. Content of defective viral genomes and standard genomes in
Fraction 1. HEp2 cells were incubated with infection medium and
supplemented with 0, 1, 10, or 20 μl of purified defective viral
particles (pDP) (Fraction 1). Cells were infected with RSV-HD at an MOI
of 1, serving as a positive control. Samples were harvested at 10 h post
infection. RNA was extracted followed by DVG-RT-PCR to detect DVGs (A)
and genome qPCR to quantify the full-length viral genome content (B).
The bands observed during pDP supplementation mirrored the pattern in
RSV-HD infection. Little viral replication of standard virus was
detected during pDP supplementation, demonstrating that this fraction is
enriched in DVGs (but not genome). The four major bands were further
confirmed by sequencing [see Sun et al. (2015), Figure S2]. For details
on the RSV genome qPCR, please refer to Tapia et al. (2013).

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